1 Real-Time PCR, a tool for the analysis and quantitation of WIS2-1A retrotransposon in hulled wheat Mondini L, Porceddu E, Pagnotta MA University of Tuscia, Via S. De Lellis, 01100 Viterbo (Italy) ABSTRACT The Real-time PCR technique was adopted to assess the genetic variability present in five accessions of einkorn (T. monococcum, 2n = 2x = 14), emmer (T. turgidum, 2n = 4x = 28) and spelt (T. spelta, 2n = 6x = 42). A simple Real-time PCR assay, based on SYBRGreen I dye, was employed to detect the copy number of one of the most important retrotransposons present in Triticum genomes, WIS2-1A. It is the first retrotransposon found in wheat and was primarily observed as an insertion into a High- Molecular-Weight (HMW) storage protein gene in T. aestivum. It represents an ancient DNA element that probably was already present in the common diploid ancestor of the Triticeae tribe. In the present work, it has been developed and optimized using a Real-time PCR assay which has permitted the detection of the presence and the number of retrotransposons. Significant differences were observed in the WIS2-1A copy number both among species and among accessions within species. Furthermore, as expected, the lowest copy number was observed for T. monococcum which represents the diploid level present among hulled wheat. On the other hand, a similar number of copies has been observed in T. dicoccum (tetraploid) and in T. spelta (hexaploid). In previous studies in barley, a strong correlation between the retrotransposons copy number and genome size was observed; recently in wheat it has been demonstrated that the wheat genome A has a higher transposable element content than genomes B and D. Therefore, this work confirms previous results where it has been observed that the A ancestral genome may have under-gone differential genome expansion caused by Class I elements prior to speciation of the tetraploid wheat ancestor; hence the amount of retrotransposon is not linearly linked to the ploidy level of the wheat species. INTRODUCTION Plant retrotransposons are grouped into two distinct classes. Class I, commonly referred to as retroelements or retrotransposons, are characterized by the presence of a long terminal repeat (LTR) and movement via RNA intermediate. Class II elements, or LINE (Long Interspersed Nuclear Elements)-like retrotransposons, transpose by a cut-and-paste mechanism with no RNA intermediate; they have no LTR, but are flanked by terminal inverted repeats. Although transposon insertion can have deleterious effects on the host genome, transposons are considered important for adaptative evolution, and can be instrumental in the acquisition of novel traits (1) . Retrotransposons are particularly important in the Triticum species, since about 80% of the wheat genome consists of repetitive elements (2) . WIS 2-1A was the first retrotransposon to be identified in wheat (3) , in the form of an 8kbp insertion within a high- molecular-weight glutenin subunit gene (4) . It is common in hortologous sequences, and shows a high level of intergenomic but a low level of intragenomic variability (5) . Its insertion site is flanked by a 5bp duplication, with an LTR of 500bp, and its termini contain an almost identical, but inverted 6bp sequence, beginning with TG and ending with CA. WIS 2-1A belongs to the Ty1-copia family, but has lost its capacity to transpose autonomously, as a result of the accumulation of mutations (6) . In addition, it appears to have no specific insertion target, or at least, its stringency is too low to prevent the insertion into other parts of the genome. WIS 2-1A is only rarely trans- activated in vivo, and is clearly an ancient DNA element, probably already present in the diploid progenitor of the Triticeae tribe (5) . The copy number of WIS 2-1A has been estimated to be about 200 per haploid genome in bread wheat (5) . The detection of variation in retrotransposon copy number to date has been achieved by assessing signal intensity either at the endpoint of a PCR reaction or in restriction digests, or by PCR- ELISA. The latter represents the standard tool for the quantification and analysis of gene expression (7) . The development of real time PCR has made possible the direct monitoring of the amplification reaction during its progress. We present here the real time PCR protocol optimization and its utilization for quantifying retrotransposons copy number in the three ploidy levels of hulled wheat. MATERIALS AND METHODS Genomic DNA was isolated, following the Dvorak et al. (8) procedure, from leaves of five accessions of each of the three hulled wheat species, einkorn (T. monococcum, 2n = 2x = 14), emmer (T. turgidum, 2n = 4x = 28) and spelt (T. spelta, 2n = 6x = 42) (Table 1). Both quality and concentration of the DNA were assessed by agarose gel electrophoresis and spectrophotometry. Functional primer pairs were generated to amplify fragments of WIS 2-1A (5) (5'-AAGAAAGGTTGT ATGTGATA-3', 5'-GTCAACAACATATACTCATC-3'. Reactions were made up to a final volume of 10µl, containing 10ng template DNA, 0.6µM of each primer, 100µM dNTP, 5mM MgCl 2 and 0.5U Taq DNA polymerase. The amplification regime consisted of an initial denaturation step of 95°C/2min, followed by 45 cycles of 94°C/1min, 52°C/2min and 72°C/2min, ending with an extension step of 72°C/10min. PCR products were separated by both agarose and polyacrylamide gel electrophoresis,